Analysis of competence in cultured sea urchin micromeres.

Sea urchin embryo micromeres form the primary mesenchyme, the skeleton-producing cells of the embryo. Almost nothing is known about nature and timing of the embryonic cues which induce or initiate spicule formation by these cells. A related question concerns the competence of the micromeres to respond to the cues. To examine competence in this system we have exposed cultured sea urchin micromeres to an inducing medium containing horse serum for various periods of time and have identified a period when micromeres are competent to respond to serum and form spicules. This window, between 30 and 50 h after fertilization, corresponds to the time when mesenchyme cells in vivo are aggregating and beginning to form the syncytium in which the spicule will be deposited. The loss of competence after 50 h is not due to impaired cell health since protein synthesis at this time is not significantly different from controls. Likewise the accumulation of a spicule matrix mRNA (SM 50) and a cell surface glycoprotein (msp 130), both indices of micromere/mesenchyme differentiation, still occurs in cells that have lost competence to respond to serum by forming spicules. These experiments demonstrate that the acquisition and loss of competence in these cells are regulated developmental events and establish an in vitro system for the identification of the molecular basis for inductive signal recognition and signal transduction.     

Mass isolation and culture of sea urchin micromeres

Summary A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of >99% pure, viable micromeres (2.3 to 4.6 × 10⁸ cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development. This work was supported in part by the following grants from the National Institutes of Health: Grant HL-10312 to A.H.W., Grant GM-20784 to Helen R. Whiteley, Grant ES-02190 to N. Karle Mottet, M.D., and Training Grants ES-07032 and HD-00266.     

The onset of collagen synthesis in sea urchin embryos

In Arbacia punctulata and Strongylocentrotus purpuratus, two species of sea urchins, collagen synthesis begins during gastrulation and increases many-fold before reaching a plateau in the late pluteus stage. A collagen extraction method involving treatment with 0.1 M NaOH and hot 10% trichloroacetic acid provided the basis for a sensitive assay of collagen synthesis.     

Time-lapse and electron microscopic studies of sea urchin micromeres

Summary The cleavage pattern of the young sea urchin embryo was studied by means of light and electron microscopy.The micromeres, which are known to have a strong organizing effect on the embryo, were found to form a syncytium with their neighbouring micromeres and with the macromeres. The cell walls between these cells were observed to be incomplete while there were interphase nuclei with intact nuclear membranes in the micro- and the macromeres. Similar phenomena with a break down of the cell membranes were not observed between macro- and mesomeres while there were intact interphase nuclei in these cells. Micromeres implanted on macromeres or mesomeres were found to coalesce with these latter cells in the course of a few minutes. During interphase, when the nuclei of both micro- and mesomere (macromere) had intact nuclear membranes, there also was a break down of the cell walls and a syncytium was formed by the host cell and the implanted micromere (see Fig. 6).The primary mesenchyme cells, which are regarded as the descendants of the micromeres, were also studied and were likewise found to form true syncytia.The importance to embryogenesis of this unique formation of syncytia is discussed.     

The fate of the small micromeres in sea urchin development

We show that in sea urchin embryos, the daughter cells of the small micromeres become part of the coelomic sacs, in contrast to the long-held view that these sacs are purely of macromere origin. In addition, after prolonged mitotic quiescence, and following their incorporation into the coelomic sacs, these cells resume dividing, contrary to the previous view that they do not divide. Since coelomic sac cells give rise to much of the adult urchin, our results indicate that the small micromeres are founders of cell lineages involved in the formation of adult tissues. The setting aside of these cells in a nondividing state may be analogous to a phenomenon in Drosophila development, in which primordial imaginal and germ cells divide approximately once after the blastoderm stage and do not resume dividing until the larval stage.     

Collagen metabolism and spicule formation in sea urchin micromeres

The role of collagen or collagen-like protein(s) in the in vitro formation of the sea urchin embryonic skeleton was investigated using isolated micromeres of Strongylocentrotus purpuratus. Micromeres were cultured in sea water containing 4% horse serum on tissue culture plastic or an extracellular matrix of type I collagen. The effect of proline analogs and an inhibitor of collagen hydroxylation on in vitro spicule formation in both culture systems was monitored. When micromeres are cultured in the presence of proline analogs l-azetidine-2-carboxylic acid and l-3,4-dehydroproline which disrupt collagen metabolism, spicule formation is significantly less inhibited on a collagen substratum than on plastic. Culturing micromeres on plastic in the presence of α,α′-dipyridyl, an inhibitor of collagen hydroxylation, resulted in almost complete inhibition of spicule formation. The inhibition by α,α′-dipyridyl can be overcome by culturing micromeres on collagen substratum. These results do not support the idea of collagen being the calcified organic matrix of the spicule. Rather, they suggest that micromeres synthesize a collagen-like extracellular matrix which is necessary for spicule formation. Inhibition of this activity by proline analogs or a collagen processing inhibitor can be overcome by providing the cells with a previously deposited extracellular matrix.     

Developmental potential of small micromeres in sea urchin embryos

The large micromeres (lMics) of echinoid embryos are reported to have distinct potentials with regard to inducing endo-mesoderm and autonomous differentiation into skeletogenic cells. However, the developmental potential of small micromeres (sMics), the sibling of lMics, has not been clearly demonstrated. In this study we produced chimeric embryos from an animal cap recombined with various numbers of sMics, in order to investigate the developmental potential of sMics in the sea urchin Hemicentrotus pulcherrimus and the sand dollar Scaphechinus mirabilis. We found that sMics of H. pulcherrimus had weak potential for inducing presumptive ectoderm cells to form endo-mesoderm structures. The inducing potential of ten sMics was almost equivalent to that of one lMic. The sMics also had the potential to differentiate autonomously into skeletogenic cells. Conversely, the sMics of S. mirabilis did not show either inductive or skeletogenic differentiation potential. The sMics of both species had the potential to induce oral-aboral axis establishment. These results suggest that the potential for sMics to differentiate into skeletogenic cells and for inducing the presumptive ectoderm to differentiate into endomesoderm differs across species, while the potential of sMics to induce the oral-aboral axis is conserved among species.     

Small micromeres contribute to the germline in the sea urchin

Many indirect developing animals create specialized multipotent cells in early development to construct the adult body and perhaps to hold the fate of the primordial germ cells. In sea urchin embryos, small micromeres formed at the fifth division appear to be such multipotent cells: they are relatively quiescent in embryos, but contribute significantly to the coelomic sacs of the larvae, from which the major tissues of the adult rudiment are derived. These cells appear to be regulated by a conserved gene set that includes the classic germline lineage genes vasa, nanos and piwi. In vivo lineage mapping of the cells awaits genetic manipulation of the lineage, but previous research has demonstrated that the germline is not specified at the fourth division because animals are fertile even when micromeres, the parent blastomeres of small micromeres, are deleted. Here, we have deleted small micromeres at the fifth division and have raised the resultant larvae to maturity. These embryos developed normally and did not overexpress Vasa, as did embryos from a micromere deletion, implying the compensatory gene regulatory network was not activated in small micromere-deleted embryos. Adults from control and micromere-deleted embryos developed gonads and visible gametes, whereas small micromere-deleted animals formed small gonads that lacked gametes. Quantitative PCR results indicate that small micromere-deleted animals produce background levels of germ cell products, but not specifically eggs or sperm. These results suggest that germline specification depends on the small micromeres, either directly as lineage products, or indirectly by signaling mechanisms emanating from the small micromeres or their descendants.     

Serum effects on the in vitro differentiation of sea urchin micromeres

When micromeres isolated from the 16-cell stage of Strongylocentrotus purpuratus are cultured in sea water containing 3.5% horse serum, they produce spicules at approximately the same time as in normal development. The serum requirement of the micromeres has been investigated by adding serum at varying intervals after isolation or by pulsing the cells with serum at specific times during their in vitro development. The optimum time of serum addition for spicule formation is 36 h after fertilization (AF). Further delay in the addition of serum results in a reduction in the number of spicules formed in culture and a delay in the time at which they appear. A 1-h pulse of serum at 36 h AF is sufficient to initiate a response in some of the micromere aggregates. A 12-h pulse at 36 h AF produces the maximum number of spicules per culture. The critical period for serum addition, 36-48 h AF, corresponds to the time in the normal embryo at which the syncytial primary mesenchyme ring is formed. Electron micrographs of cultured cells demonstrate that micromeres cultured without serum until 48 h AF fail to form pseudopodial extensions and remain as rosette-like clusters of cells. If serum is present, extensive pseudopodial networks form which resemble the primary ring syncytium. These results suggest that serum acts to stimulate fused pseudopodial networks in cultures of micromeres and that the resulting syncytium is necessary for spicule formation.     

Cleavage and differentiation in the sea urchin embryo transplantation studies of micromeres

Summary Micromeres isolated from the 16-cell stage were implanted on mesomeres or macromeres from the same larva. The process of coalescence and the cleavage pattern of the transplanted micromeres were studied by means of light and electron microscopy.The transplanted micromere shows the same cleavage pattern as the micromerein situ. A close contact is established between the micromere and the host cell and cytoplasmic bridges are found between the cells.The micromere is dependent on its adjoining blastomere(s) and the rate of cleavage is slowed down when the micromere is isolated. Macromeres and mesomeres are not subjected to a similar change in rate of cleavage when isolated from the rest of the embryo.The ratio mitochondria/yolk in micromeres is different from that observed in macro- or mesomeres and the possible consequences of this fact are discussed.     

Mechanisms of calcium elevation in the micromeres of sea urchin embryos

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.     

Limited complexity of the RNA in micromeres of sixteen-cell sea urchin embryos

The sequence complexity of sea urchin embryo micromere RNA is about 75% of that of total 16-cell embryo cytoplasmic RNA, as reported earlier by Rodgers and Gross [Rodgers, W. H., and Gross, P. R. (1978) Cell, 14, 279–288]. In contrast to the rest of the embryo, there are few, if any, complex maternal RNA species in the micromere cytoplasm which are not represented in the polysomes. The micromeres do not contain detectable quantities of high-complexity nuclear RNA, though such RNA exists in other cells of the fourth-cleavage embryo.     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Wheat germ agglutinin binding to the micromeres and primary mesenchyme cells of sea urchin embryos

Fluorescein isothiocyanate-conjugated wheat germ agglutinin (WGA-FITC) binds exclusively to the primary mesenchyme cells when the lectin is microinjected into the blastocoels of living Lytechinus pictus and Strongylocentrotus droebachiensis embryos. WGA-FITC binding increases throughout the period of primary mesenchyme cell migration and aggregation. Similar binding is observed in embryos cultured in sulfate-free seawater (SFSW) but not in seawater (ASW) containing tunicamycin. The temporal expression of WGA-FITC binding sites in vivo is also correlated with the pattern of binding observed in vitro. Sixteen-cell stage Arbacia punctulata embryos were dissociated in Ca²⁺ and Mg²⁺-free seawater (CMFSW) and the micromeres isolated using sucrose gradients. Arbacia micromeres, cultured in ASW containing calf serum, first bind WGA-FITC during the period when primary mesenchyme cell ingression occurs in control embryos. Micromeres cultured in the presence of tunicamycin do not develop WGA binding sites. The temporal expression of WGA-FITC binding in micromere cultures is unaffected by the absence of sulfate, but the size and morphology of aggregates cultured in SFSW differ from that of the controls.